JP2023523372A - Antibody-drug conjugates and preparations thereof - Google Patents

Abstract

Anti-CD20 antibody-drug conjugates, preparations comprising the antibody-drug conjugates, compositions comprising the antibody-drug conjugates, and pharmaceutical uses of the antibody-drug conjugates are provided.

Description

The present invention relates to the field of biomedicine, in particular to antibody-drug conjugates, preparations comprising antibody-drug conjugates, compositions comprising antibody-drug conjugates, and pharmaceutical uses of antibody-drug conjugates.

Currently, treatments for non-Hodgkin's lymphoma (NHL) include conventional surgery, chemotherapy, radiation therapy, bone marrow or hematopoietic stem cell transplantation, immunotherapy and targeted therapy. Although conventional chemotherapy has some curative effect on NHL patients, it has severe systemic side effects, poor tolerance, and easy recurrence, limiting its application to patients. Targeted drugs, especially antibody drugs, have received a great deal of attention due to their remarkable curative efficacy and minimal systemic side effects during therapeutic intervention.

In recent years, many CD20-targeted monoclonal antibodies (eg, Rituximab, Rituxan®) have been developed and their anti-tumor activity has been validated in clinical trials.

However, with the deepening of clinical application, the limitations of rituximab treatment are becoming increasingly apparent, namely that there are patients with primary and acquired resistance to rituximab during the treatment process, resulting in a decrease in the therapeutic effect of rituximab. or becomes invalid. In addition, existing CD20-targeted monoclonal antibodies are not ideal in terms of efficacy to treat tumors and require concomitant use with chemotherapeutic agents to improve anti-tumor efficacy, which undoubtedly increases the risk of toxicity. Let In this regard, commercially available CD20-targeted monoclonal antibodies, typified by Rituximab, are far from meeting the need in clinical practice for the treatment of relapsed or refractory B-cell NHL (such as DLBCL and FL).

Considering the above-mentioned problems of existing CD20-targeting monoclonal antibodies, the main objective of developing CD20-targeting ADC drug ADC-1 is to treat it with better efficacy, lower toxicity and side effects, and less drug resistance. to provide a method.

Contents of the present invention The present inventors have prepared anti-CD20 antibody-drug conjugates through many experiments and creative studies and confirmed that they have good biological activity and formulation stability, thereby presenting the present invention. completed.

To this end, in a first aspect of the invention, the invention provides a structure represented by formula I:
Ab-(LD) _p Formula I
providing an antibody-drug conjugate having
Ab stands for anti-CD20 monoclonal antibody, anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or its biosimilars;
D is a cytotoxic agent, the cytotoxic agent is monomethylauristatin E (MMAE);
L represents a linker for linking the anti-CD20 monoclonal antibody with the cytotoxic agent, the linker being 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB);
p is between 3.6 and 4.0.

In some embodiments, p is 3.7-3.9.
In some embodiments, p is 3.8.

In some embodiments, LD of Formula I is vc-MMAE and has the structure shown in the following formulae.

In some embodiments, the structure of the antibody-drug conjugate has the structure shown in the formula below.

During the ceremony,
Ab represents an anti-CD20 monoclonal antibody, which is an antibody that targets CD20, such as rituximab or its biosimilars.
p is between 3.6 and 4.0.

In some embodiments, p is 3.7-3.9.
In some embodiments, p is 3.8.

In a second aspect of the invention, the invention provides an antibody-drug conjugate preparation comprising:
1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 13 mg/mL mL, 15 mg/mL, 17 mg/mL, 19 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, or 60 mg/mL ) of the antibody-drug conjugate;
concentrations of 5-35 mM (eg 5 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 25 mM, 30 mM or 35 mM) and 5.0-6.2 (eg 5.0, 5.1, Histidine buffer with a pH of 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1-6.2) liquid;
sucrose having a concentration of 2% to 10% (for example 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%);
0.01% to 0.1% (for example, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.031%, 0.032%, 0.033% %, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.04%, 0.045%, 0.05%, 0.055 %, 0.06%, 0.065%, 0.07%, 0.08%, 0.09% or 0.1%);
The antibody-drug conjugate has the structure shown in Formula II:
Ab-(LD) _q Formula II
and where
Ab stands for anti-CD20 monoclonal antibody, anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or its biosimilars;
D is a cytotoxic agent, the cytotoxic agent is monomethylauristatin E (MMAE);
L represents a linker for linking the anti-CD20 monoclonal antibody with the cytotoxic agent, the linker being 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB);
q is 3.3 to 4.3 (e.g., 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4 .2 or 4.3).

In some embodiments, q is 3.6-4.0.
In some embodiments, q is 3.7-3.9.
In some embodiments, q is 3.8.
In some embodiments, the antibody-drug conjugate has a concentration of 1-20 mg/mL.
In some embodiments, the antibody-drug conjugate has a concentration of 1-10 mg/mL.

In some embodiments, the antibody-drug conjugate has a concentration of 3-7 mg/mL.
In some embodiments, the antibody-drug conjugate has a concentration of 5 mg/mL.
In some embodiments, the histidine buffer has a concentration of 5-15 mM.
In some embodiments, the histidine buffer has a concentration of 8-12 mM.
In some embodiments, the histidine buffer has a concentration of 10 mM.

In some embodiments, the histidine buffer has a pH of 5.8-6.2.
In some embodiments, the histidine buffer has a pH of 5.6-6.0.
In some embodiments, the histidine buffer has a pH of 5.4-6.0.
In some embodiments, the histidine buffer has a pH of 5.4-6.2.
In some embodiments, sucrose has a concentration of 3%-9%.

In some embodiments, sucrose has a concentration of 4%-8%.
In some embodiments, sucrose has a concentration of 5%-7%.
In some embodiments, sucrose has a concentration of 6%.
In some embodiments, Tween-80 has a concentration of 0.02%-0.06%.
In some embodiments, Tween-80 has a concentration of 0.02%-0.05%.

In some embodiments, Tween-80 has a concentration of 0.03%-0.04%.
In some embodiments, Tween-80 has a concentration of 0.035%.

In some embodiments, the preparation includes:
an antibody-drug conjugate having a concentration of 5 mg/mL;
a histidine buffer with a concentration of 10 mM and a pH of 5.8;
6% sucrose; and 0.035% Tween-80
including.

Note that "2-10% sucrose" represents the mass-volume concentration (w/v%), meaning that the sucrose mass per 1000 mL of the antibody-drug conjugate preparation is 20-100 g, and "0.01- 0.1% Tween-80” represents mass volume concentration (w/v %) and its meaning can be understood in the same way as “2-10% sucrose” above.

Further, the concentration of "histidine buffer" refers to the concentrations of histidine and histidine hydrochloride, the pH of which is obtained by adjusting the ratio of histidine to histidine hydrochloride.

In some embodiments, when the pH is 5.0-6.2 (eg, 5.6-6.0), the antibody-drug conjugate preparation has a pH of 25±2° C./60%±5% Stable for 6 weeks under RH conditions.

In some embodiments, when the pH is 5.0-6.2 (eg, 5.6-6.0), the antibody-drug conjugate preparation is left at 25° C. for 6 weeks to Appears colorless and transparent.

In some embodiments, the antibody-drug conjugate preparation is left at 25° C. for 6 weeks when the pH is 5.0-6.2 (eg, 5.6-6.0). and no change in pH.

In some embodiments, the antibody-drug conjugate preparation is left at 25° C. for 6 weeks when the pH is 5.0-6.2 (eg, 5.6-6.0). , the density does not change.

In some embodiments, when the pH is 5.0-6.2 (eg, 5.6-6.0), the antibody-drug conjugate preparation exhibits a 2% pH when left at 25° C. for 6 weeks. SEC monomer % content reduction of less than (or less than 1%).

In some embodiments, when the pH is 5.0-6.2 (eg, 5.6-6.0), the antibody-drug conjugate preparation is left at 25° C. for 6 weeks to It has a SEC polymer % content increase of 2% or less (or 1% or less).

In some embodiments, the antibody-drug conjugate preparation is left at 25° C. for 6 weeks when the pH is 5.0-6.2 (eg, 5.6-6.0). and have no significant change in DAR values.

In some embodiments, when the pH is 5.0-6.2 (eg, 5.6-6.0), the antibody-drug conjugate preparation is left at 40° C. for 4 weeks to It is colorless and transparent in appearance.

In some embodiments, when the pH is 5.0-6.2 (eg, 5.6-6.0), the antibody-drug conjugate preparation, when left at 40° C. for 4 weeks, No change in pH.

In some embodiments, when the pH is 5.0-6.2 (eg, 5.6-6.0), the antibody-drug conjugate preparation is left at 40° C. for 4 weeks to Concentration does not change.

In some embodiments, when the pH is between 5.0 and 6.2 (eg, between 5.6 and 6.0), the antibody-drug conjugate preparation has a 5% SEC monomer % content reduction of less than (or less than 4%).

In some embodiments, when the pH is 5.0-6.2 (eg, 5.6-6.0), the antibody-drug conjugate preparation is left at 40° C. for 4 weeks to It has an SEC polymer % content increase of less than 5% (or less than 4%).

In some embodiments, when the pH is 5.0-6.2 (eg, 5.6-6.0), the antibody-drug conjugate preparation is left at 40° C. for 4 weeks to It has no significant change in DAR values.

"No significant change" or "no change" means no statistically significant change.

Furthermore, it should be noted that the above-mentioned "antibody-drug conjugate" refers to compositions containing ADC molecules with the same or different DAR values.

Specifically, the present invention provides compositions comprising multiple anti-CD20 ADC molecules. In some examples, each ADC in the compositions described herein contains the same number of one or more drug molecules. In other examples, each ADC in the compositions described herein contains a different number of one or more drug molecules.

In the antibody drug conjugates described herein, each anti-CD20 antibody has 1, 2, 3, 4, 5, 6, 7, 8 or more (preferably 2, 4, 6 or 8, more preferably 2 or 4) drug molecules can be conjugated.

The drug-antibody ratio (DAR) above refers to the number of molecules of anticancer drug bound to the anti-CD20 antibody. The number of molecules of the anticancer drug contained in the ADC described herein is usually an integer, and the number of molecules of the anticancer drug contained in the ADC described herein (e.g., formula I When p in or q) in Formula II is fraction, fraction refers to the average number of molecules of anti-cancer agent bound to each anti-CD20 antibody in a composition comprising multiple ADC molecules.

In a third aspect of the invention, the invention provides a method of preparing said antibody-drug conjugate preparation, comprising:
A reduction reaction of the anti-CD20 monoclonal antibody is performed with a reducing agent to obtain a reduced anti-CD20 monoclonal antibody, where the anti-CD20 monoclonal antibody is any antibody that targets CD20 (e.g., rituximab or its biosimilar);
performing a coupling reaction between the reduced anti-CD20 monoclonal antibody and vc-MMAE;
terminating the coupling reaction;
The quenched coupling reaction products are allowed to undergo buffer displacement to obtain an antibody-drug conjugate preparation, the antibody-drug conjugate preparation comprising:
1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 13 mg/mL mL, 15 mg/mL, 17 mg/mL, 19 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, or 60 mg/mL ) of the antibody-drug conjugate;
concentrations of 5-35 mM (eg 5 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 25 mM, 30 mM or 35 mM) and 5.0-6.2 (eg 5.0, 5.1, Histidine buffer with a pH of 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1-6.2) liquid;
sucrose having a concentration of 2% to 10% (for example 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%);
0.01% to 0.1% (for example, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.031%, 0.032%, 0.033% %, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.04%, 0.045%, 0.05%, 0.055 %, 0.06%, 0.065%, 0.07%, 0.08%, 0.09% or 0.1%).

In some embodiments, the antibody-drug conjugate has a concentration of 1-20 mg/mL.
In some embodiments, the antibody-drug conjugate has a concentration of 1-10 mg/mL.
In some embodiments, the antibody-drug conjugate has a concentration of 3-7 mg/mL.
In some embodiments, the antibody-drug conjugate has a concentration of 5 mg/mL.
In some embodiments, the histidine buffer has a concentration of 5-15 mM.

In some embodiments, the histidine buffer has a concentration of 8-12 mM.
In some embodiments, the histidine buffer has a concentration of 10 mM.
In some embodiments, the histidine buffer has a pH of 5.8-6.2.
In some embodiments, the histidine buffer has a pH of 5.6-6.0.
In some embodiments, the histidine buffer has a pH of 5.4-6.0.

In some embodiments, the histidine buffer has a pH of 5.4-6.2.
In some embodiments, sucrose has a concentration of 3%-9%.
In some embodiments, sucrose has a concentration of 4%-8%.
In some embodiments, sucrose has a concentration of 5%-7%.
In some embodiments, sucrose has a concentration of 6%.

In some embodiments, Tween-80 has a concentration of 0.02%-0.06%.
In some embodiments, Tween-80 has a concentration of 0.02%-0.05%.
In some embodiments, Tween-80 has a concentration of 0.03%-0.04%.
In some embodiments, Tween-80 has a concentration of 0.035%.
In some embodiments, the reducing agent is DTT.

In some embodiments, the method of preparation comprises
1) Collect 10 mg of anti-CD20 monoclonal antibody and replace with reducing buffer (25 mM sodium borate, pH 8.0, 25 mM NaCl, 5 mM EDTA) using a 15 mL 30 KD ultrafiltration device for a total of 3 replacements. to obtain a final volume of about 1 mL, transfer (weigh) the resulting solution to a new Eppendorf centrifuge tube, detect protein concentration and calculate total protein;
2) Add 2.0 to 3.0 times the moles of DTT to the antibody, incubate at room temperature for 2 hours, mix continuously, and then remove coupling buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 5 mM EDTA), substituted three times, collected the concentrated solution, detected the protein concentration at A280 (absorbance at a wavelength of 280 nm), weighed, determined the total protein amount, A sample of 10 μL is collected, and the number of free sulfhydryl groups is determined by the Ellman method,
The molar concentration of free sulfhydryl groups is calculated by the following formula (where A412 represents the absorbance at a wavelength of 412 nm):

b: optical path length of the cuvette (usually 1 cm)
The number of moles of free sulfhydryl groups should be calculated according to the molar concentration of free sulfhydryl groups and the volume of the total protein solution;
3) vc-MMAE (dissolved in DMSO) is added to the reduced antibody at 1.0-1.5 times the number of moles of free sulfhydryl groups, mixed well, then at room temperature for 2 hours. , react with intermittent stirring; add N-acetylcysteine to the reaction system at 20 times the number of moles of vc-MMAE to the reaction solution, mix well, and leave for 5 minutes;
4) Replace the solution obtained in step 3 with the conjugate stock solution (10 mM histidine, 6% sucrose, 0.035% PS80, pH 5.8) using a 15 mL 30 KD ultrafiltration device, for a total of 3 times. to obtain antibody-drug conjugate preparations and store at 4°C.

In a fourth aspect of the invention, the invention provides a composition comprising said antibody-drug conjugate, or said antibody-drug conjugate preparation, or an antibody-drug conjugate preparation prepared by said method. do.

In some embodiments, the composition further comprises at least one pharmaceutically acceptable carrier, diluent or excipient.

In some embodiments, the composition further comprises known chemotherapeutic agents for the treatment of tumors, such as doxorubicin (Adriamycin), cyclophosphamide and taxanes [paclitaxel (Taxol) and Docetaxel (Taxotere)], Capecitabine (Xeloda), Gemcitabine (Gemzar), Vinorelbine (Navelbine), Tamoxifen, Aromatase Inhibitors (Arimidex, Femara, Aromasin), 5-FU/Leucovorin, Irinotecan (Camptosar), Oxaliplatin, Cisplatin , carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or combinations thereof.

In some embodiments, the composition comprises (1) glucocorticoids such as cortisone and prednisone; (2) microbial metabolites such as cyclosporine and tacrolimus; products; (4) polyclonal and monoclonal anti-lymphocyte antibodies such as anti-lymphocyte globulin and OKT3; (5) alkylating agents such as cyclophosphamide. Specifically, immunosuppressants include, for example, methylprednisolone, prednisone, azathioprine, Prograf, Zenapax, Simulect, cyclosporine, tacrolimus, rapamycin, mycophenolic acid, mizoribine, cyclophosphamide, fingolimod, and the like.

In a fifth aspect of the present invention, the present invention provides said antibody-drug conjugate or said antibody-drug conjugate preparation in the manufacture of a medicament for preventing and/or treating CD20-expressing cancers or immune diseases. , use of said antibody-drug conjugate preparation prepared by said method, or said composition.

In some embodiments, the CD20-expressing cancer is lymphoma or leukemia.

In some embodiments, the lymphoma is non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, small lymphocytic lymphoma, or diffuse large B-cell lymphoma.

In some embodiments, the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, B-cell prolymphocytic leukemia, or acute lymphocytic leukemia.

In some embodiments, the CD20-expressing immune disease is rheumatoid arthritis, granulomatosis with polyangiitis, Wegener's granulomatosis, microscopic polyangiitis, or multiple sclerosis.

In some embodiments, the rheumatoid arthritis is severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.

Preferably, in some embodiments, the CD20-expressing cancer is CD20-positive B-cell lymphoma.

In some embodiments, the CD20-positive B-cell lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant CD20-positive B-cell lymphoma.

In some embodiments, the lymphoma is non-Hodgkin's lymphoma.

In some embodiments, the lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant non-Hodgkin's lymphoma.

In some embodiments, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.

In some embodiments, the non-Hodgkin's lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant diffuse large B-cell lymphoma.

In a sixth aspect of the present invention, the present invention provides a method of preventing and/or treating CD20-expressing cancer or immune disease, wherein a subject in need thereof is treated with a prophylactically and/or therapeutically effective administering an amount of said antibody-drug conjugate, or said antibody-drug conjugate preparation, or said antibody-drug conjugate preparation prepared by said method, or said composition.

In some embodiments, the CD20-expressing cancer is lymphoma or leukemia.

In some embodiments, the lymphoma is non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, small lymphocytic lymphoma, or diffuse large B-cell lymphoma.

In some embodiments, the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, B-cell prolymphocytic leukemia, or acute lymphocytic leukemia.

In some embodiments, the CD20-expressing immune disease is rheumatoid arthritis, granulomatosis with polyangiitis, Wegener's granulomatosis, microscopic polyangiitis, or multiple sclerosis.

In some embodiments, the rheumatoid arthritis is severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.

In some embodiments, the CD20-expressing cancer is preferably CD20-positive B-cell lymphoma.

In some embodiments, the CD20-positive B-cell lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant CD20-positive B-cell lymphoma.

In some embodiments, the lymphoma is non-Hodgkin's lymphoma.

In some embodiments, the lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant non-Hodgkin's lymphoma.

In some embodiments, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.

In some embodiments, the non-Hodgkin's lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant diffuse large B-cell lymphoma.

In some embodiments, where the subject is an NHL PDX model, the dose administered to a subject in need thereof is 0.3-10 mg/kg (e.g., 0.3 mg/kg, 1 mg/kg, 3 mg/kg /kg, 10 mg/kg).

In some embodiments, when the subject is an NHL PDX model, the dose administered to a subject in need thereof is 0.3-3 mg/kg, 1-3 mg/kg, 1-10 mg/kg, or 3-10 mg/kg.

In some embodiments, when the subject is human, the dose administered to a subject in need thereof is 0.1-5 mg/kg (eg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg /kg, 3 mg/kg or 5 mg/kg).

In some embodiments, a prophylactically and/or therapeutically effective amount of the aforementioned antibody-drug conjugates, or the aforementioned antibody-drug conjugate preparations, or the aforementioned antibody-drug conjugates prepared by the aforementioned methods. A gating preparation, or the aforementioned composition, is administered to a subject in need thereof once every 1, 2, 3, 4, 5, or 6 weeks.

In some embodiments, a prophylactically and/or therapeutically effective amount of said antibody-drug conjugate or said antibody-drug conjugate preparation prepared by said method or antibody prepared by said method - The drug conjugate preparation, or said composition, is administered to a subject in need thereof once every 18-24 days.

In some embodiments, the method further comprises administering an additional chemotherapeutic or immunosuppressive agent for treatment of the tumor to the subject in need thereof.

In some embodiments, the chemotherapeutic agent is, for example, doxorubicin (Adriamycin), cyclophosphamide and taxanes [paclitaxel (Taxol) and docetaxel (Taxotere)], capecitabine (Xeloda), gemcitabine (Gemzar) , vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Femara, Aromasin), 5-FU/leucovorin, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone , vincristine (Oncovin), doxorubicin, prednisone, etc., or combinations thereof.

In some embodiments, the immunosuppressive agent is (1) glucocorticoids such as cortisone and prednisone; (2) microbial metabolites such as cyclosporine and tacrolimus; (3) antimicrobials such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies such as anti-lymphocyte globulin and OKT3; (5) alkylating agents such as cyclophosphamide. Specifically, immunosuppressants include, for example, methylprednisolone, prednisone, azathioprine, Prograf, Zenapax, Simulect, cyclosporine, tacrolimus, rapamycin, mycophenolic acid, mizoribine, cyclophosphamide, fingolimod, and the like.

In a seventh aspect of the invention, the invention provides said antibody-drug conjugate, or said antibody-drug conjugate preparation, or The antibody-drug conjugate preparation prepared by the method, or the composition is provided.

In some embodiments, the CD20-expressing cancer is lymphoma or leukemia.

In some embodiments, the lymphoma is non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, small lymphocytic lymphoma, or diffuse large B-cell lymphoma.

In some embodiments, the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, B-cell prolymphocytic leukemia, or acute lymphocytic leukemia.

In some embodiments, the CD20-expressing immune disease is rheumatoid arthritis, granulomatosis with polyangiitis, Wegener's granulomatosis, microscopic polyangiitis, or multiple sclerosis.

In some embodiments, the rheumatoid arthritis is severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.

In some embodiments, the CD20-expressing cancer is preferably CD20-positive B-cell lymphoma.

In some embodiments, the CD20-positive B-cell lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant CD20-positive B-cell lymphoma.

In some embodiments, the lymphoma is non-Hodgkin's lymphoma.

In some embodiments, the lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant non-Hodgkin's lymphoma.

In some embodiments, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.

In some embodiments, the non-Hodgkin's lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant diffuse large B-cell lymphoma.

In some embodiments, when the subject is an NHL PDX model, the dose administered is 0.3-10 mg/kg (eg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg) is.

In some embodiments, when the subject is an NHL PDX model, the dose administered is 0.3-3 mg/kg, 1-3 mg/kg, 1-10 mg/kg, or 3-10 mg/kg. .

In some embodiments, when the subject is a human, the dose administered is 0.1-5 mg/kg (eg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 5 mg/kg).

Beneficial Effects 1 . ADC-1 is an ADC formed by coupling the rituximab biosimilar MAB801 with vc-MMAE, binds to CD20 receptors on the surface of tumor cells, undergoes endocytosis and release of MMAE; It kills CD20-positive non-Hodgkin's lymphoma (NHL) cells by thereby controlling tumor growth and causing tumor regression.
2. ADC-1 exhibits significant pharmacodynamic effects in inhibiting tumor cell growth in various CD20-expressing NHL cell lines and in various CD20-expressing human NHL PDX tumor models; TM) also shows a significant inhibitory effect on tumor cell proliferation in the resistant NHL PDX model.
3. The ADC-1 preparation process is superior to other preparation processes by simply completing the entire reaction step sequentially in one vessel without intermediate purification steps.
4. The process for preparing ADC-1 requires only one ultrafiltration purification step for the entire process and does not require a chromatographic step, which is superior to other preparative steps.
5. ADC-1 has good formulation stability.

FIG. 1 shows the HIC-HPLC spectrum of ADC-1 of an example of the present invention. FIG. 2 shows representative cell-killing action curves of ADC-1 and Rituxan® in Daudi cell lines according to examples of the present invention, where inhibition represents percent inhibition. FIG. 3 shows representative cell-killing action curves of ADC-1 and Rituxan® in the Jeko-1 cell line according to examples of the present invention, representing percent inhibition. FIG. 4 shows representative cell-killing action curves of ADC-1 and Rituxan® in Raji cell lines according to examples of the present invention, where inhibition represents percent inhibition. FIG. 5 shows representative cell-killing action curves of ADC-1 and Rituxan® in Ramos cell lines according to examples of the present invention, where inhibition represents percent inhibition. FIG. 6 shows a schematic representation of the effect of ADC-1 and RITUXAN® on tumor volume of CD20-positive human lymphoma (NHL) PDX model LYM#004 according to an example of the present invention. FIG. 7 shows a schematic representation of the effect of ADC-1 and Rituxan® on tumor volume of CD20-positive human lymphoma (NHL) PDX model LYM#013 according to an example of the present invention. FIG. 8 shows a schematic representation of the effect of ADC-1 and Rituxan® on tumor volume of CD20-positive human lymphoma (NHL) PDX model LYM#016 according to an example of the present invention. Figure 9 shows a schematic representation of the effect of pH on the SEC stability of the active substance ADC-1 (10 mM histidine buffer system), ADC-1 in a 10 mM histidine system at different pHs, left panel at 25°C. The figure on the right shows the trend of SEC polymer content of ADC-1 after 6 weeks at 25°C and 4 weeks at 40°C. .

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION Embodiments of the present invention are described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only for illustrating the invention. It will be understood that they should not be used and should be construed as limiting the scope of the invention. If specific conditions are not described in the examples, the conditions are conventional or according to the conditions presented by the manufacturer. Reagents or instruments used without manufacturer's instructions are conventional commercially available products.

In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In addition, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are terms and routine procedures widely used in the corresponding fields. . Meanwhile, definitions and explanations of related terms are provided below for a better understanding of the present invention.

In the present invention, unless otherwise specified, any concentration range, percentage range, ratio range or numerical range is understood to include any integer value within the stated range and, where appropriate, any fractional value within the range. It should be.

In the present invention, the term "antibody" generally refers to an immunoglobulin molecule composed of two identical pairs of polypeptide chains, each pair consisting of one "light" (L) chain and one "heavy" (H) chain. have chains. Antibody light chains are divided into two categories, kappa and lambda. Heavy chains are classified into five types, μ, δ, γ, α, and ε, and antibodies are classified into five types, IgM, IgD, IgG, IgA, and IgE, depending on the heavy chain. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and heavy chains also include a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies are capable of mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and component C1q of the complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy/light chain pair each form the antibody binding site. The assignment of amino acids to regions or domains follows the Kabat sequence definitions for proteins of immunological interest (National Institutes of Health, Bethesda, Md. (1987 and 1991), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

Antibodies suitable for use in the present invention are antibodies that target CD20, and anti-CD20 monoclonal antibodies are any antibodies that target CD20, such as rituximab or its biosimilars. Of these, biosimilars refer to antibody products that have the same sequences, the same physicochemical properties and biological activities as rituximab, and the same clinical safety and efficacy as rituximab.

In the present invention, the structure of MMAE is as follows.

In the present invention, the drug:antibody ratio (DAR) or drug loading (loading) can be either p or q, i.e. whole numbers or fractions, Formula I: Ab-(LD)p or Formula II : represented by the average number of drug modules (ie, cytotoxic agents) per antibody in a molecule of Ab-(LD)q. ADCs of formula I comprise a collection of antibodies bound to drug modules ranging from (3.6-4.0) and ADCs of formula II bound to drug modules ranging from (3.3-4.3) contains a collection of generated antibodies. The average number of drug modules per antibody in ADC preparations from conjugation reactions can be confirmed by conventional means such as mass spectroscopy, ELISA assays, HIC and HPLC. A quantitative distribution of ADC with respect to p or q can also be determined. Separation, purification and validation of homogeneous ADCs, optionally with p or q at a value from ADCs with other drug loads, can be achieved by means such as reverse phase HPLC or electrophoresis.

In some embodiments, less than the theoretical maximum amount of drug module is attached to the antibody in the conjugation reaction. In general, antibodies do not contain many free and reactive cysteine thiol groups with which drug modules can be attached; indeed, the majority of cysteine thiol groups in antibodies exist as disulfide bridges. In some embodiments, the antibody is treated with a reducing agent, such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine ( TCEP).

The antibody-drug conjugates of the invention can be used in combination with known chemotherapeutic agents or immunosuppressive agents for the treatment of tumors, such as doxorubicin (adriamycin), cyclophosphamide and taxanes [paclitaxel (Taxol) and docetaxel (Taxotere)], capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Femara, Aromasin), 5-FU/leucovorin, irinotecan ( camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or combinations thereof; (2) microbial metabolites such as cyclosporine and tacrolimus; (3) antimetabolites such as azathioprine and 6-mercaptopurine; (4) antilymphocyte globulin and OKT3 such as OKT3; polyclonal and monoclonal anti-lymphocyte antibodies; (5) alkylating agents such as cyclophosphamide. Specifically, immunosuppressants include, for example, methylprednisolone, prednisone, azathioprine, Prograf, Zenapax, Simulect, cyclosporine, tacrolimus, rapamycin, mycophenolic acid, mizoribine, cyclophosphamide, fingolimod, and the like.

In the present invention, "treatment" refers to clinical intervention that attempts to alter the natural course of the individual or cells being treated, either for prophylaxis or in the course of clinical pathology. Desirable effects of treatment include prevention of disease onset or recurrence, relief of symptoms, attenuation of direct or indirect pathological consequences of disease, prevention of metastasis, slowing the rate of disease progression, amelioration or reduction of disease state. , and alleviation or improvement of prognosis. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used to delay the onset of the disease or disorder or delay the progression of the disease or disorder. The above parameters used to assess treatment success and disease improvement can be readily measured by routine procedures familiar to physicians. For cancer therapy, efficacy can be measured, for example, by assessing time to disease progression (TTP) and/or by determining response rate (RR).

In the present invention, "subject" refers to vertebrate animals. In certain embodiments, vertebrate refers to mammals. Mammals include, but are not limited to, farm animals (eg, cows), pets (eg, cats, dogs, and horses), primates, mice, and rats. In some embodiments, mammal refers to humans.

As used herein, an "effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic effect. A "therapeutically effective amount" of a substance/molecule of the invention may vary depending on factors such as the condition, the age, sex and weight of the individual, and the ability of the substance/molecule to elicit a desired response in the individual. A therapeutically effective amount also includes an amount in which toxic or detrimental effects of the substance/molecule are outweighed by a therapeutically beneficial effect. A "prophylactically effective amount" refers to an amount effective, at dosages and for times necessary to achieve the desired prophylactic effect. Because a prophylactic dose is administered to a subject prior to the onset of disease or at an early stage of disease, the prophylactically effective amount will usually, but not necessarily, be less than the therapeutically effective amount. be. In the case of cancer, therapeutically effective amounts of drugs reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow and preferably arrest) tumor metastasis; inhibit tumor growth to some extent; and/or reduce one or more symptoms associated with cancer to some extent.

Appropriate doses of the antibody-drug conjugates of the invention (when used alone or in combination with one or more additional therapeutic agents such as chemotherapeutic agents) for the prevention or treatment of disease is the type of disease to be treated, the type of antibody-drug conjugate, the severity and course of the disease, whether the antibody-drug conjugate is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical It will depend on history and responsiveness to antibody-drug conjugates and the judgment of the attending physician. The antibody-drug conjugate is suitably administered to the patient at one time or over a series of treatments. An exemplary dose of antibody-drug conjugate can range from about 0.1 mg/kg to about 5 mg/kg. Accordingly, one or more of about 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, or 5 mg/kg (or any combination thereof) of the antibody-drug conjugate. A dose can be administered to the patient.

A "pharmaceutically acceptable carrier" as used in the present invention includes pharmaceutically acceptable carriers, excipients or stabilizers, which are used in the cells or mammals to which they are exposed. It is non-toxic at the doses and concentrations used. Typically, physiologically acceptable carriers are pH buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants, including, for example, ascorbic acid; low molecular weight (about 10 residues proteins such as serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and glucose, mannose, chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; Nonionic surfactants are included.

In the present invention, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass.), incorporated herein by reference.

The invention is further described below in conjunction with specific examples.

Example 1: Construction of a production cell line for the antibody MAB801 (used for the naked antibody portion of the antibody-drug conjugate ADC-1). Analysis by MS determined the amino acid sequences of the light and heavy chains of the antibody. The entire light and heavy chain gene sequences were further confirmed according to the sequence information of the patent (Anderson-1998, Patent No: US5736137A), and the light and heavy chain gene DNA sequences of MAB801 were obtained by gene synthesis, respectively, Next, the light chain (light chain, LC) and heavy chain (heavy chain, HC) gene expression plasmids were constructed separately using a double-plasmid vector expression system (from Invitrogen Company), the two plasmids A cell population co-transfected into mammalian host cell CHO DG44 (from Invitrogen Company) and co-integrated with light and heavy chain gene sequences was obtained after two-step stress screening. The cell populations were evaluated for their ability to express antibodies and were found to meet the requirements. Single cell clones were screened using semi-solid media, the resulting single cell clones were screened by fed-batch experiments to select the cloned cell line with the highest expression level of antibody, and the cells were A production cell bank of MAB801 was prepared based on the strain.

Gene sequencing was performed in a monoclonal cell line and the sequencing results showed that the sequences encoding the light and heavy chains of MAB801 were completely identical to commercially available rituximab.

Example 2: Preparation of antibody MAB801 (for the naked antibody portion of antibody-drug conjugate ADC-1)
MAB801 was prepared by a feed-batch cell culture process widely used in the antibody field. The process began with the harvesting of cell seeds for the working cell bank and consisted of the following main steps: shake flask inoculation, shake flask cell growth, stepwise cell growth culture in reactors, and for fed-batch culture. followed by purification processes including: fermentation broth clarification (depth filtration), protein A affinity chromatography, low pH virus inactivation and depth filtration, PB incubation, anion exchange chromatography, positive Filtration and packaging are performed to obtain the product through processes such as ion exchange chromatography, virus removal filtration, tangential flow ultrafiltration and diafiltration.

The detection results indicated that the biosimilarity of Rituximab produced was very similar to Rituximab in terms of molecular weight and other physical and chemical indices.

Example 3: Methods for preparing ADC-1, CD20 targeting antibody-drug conjugates 1) Take 10 mg of MAB801 antibody and filter with reducing buffer (sodium borate 25 mM, pH 8.0, 25 mM NaCl, 5 mM EDTA) for a total of 3 substitutions to a final volume of approximately 1 mL, transfer the resulting solution to a new Eppendorf centrifuge tube (weigh), detect protein concentration, determine total protein Concentration was calculated.
2) 2.5-fold molar DTT was added to the antibody, incubated for 2 hours at room temperature and mixed continuously; , pH 7.2, 150 mM NaCl, 5 mM EDTA) for a total of three substitutions. The concentrated liquid was taken, the protein concentration was measured using A280, the total protein amount was calculated by weighing, a 10 μL sample was taken, and the number of free sulfhydryl groups was measured by Ellman's method. The molar concentration of free sulfhydryl groups was calculated by the following formula.

b: optical path length of the cuvette (usually 1 cm)
The number of moles of free sulfhydryl groups was calculated based on the molar concentration of free sulfhydryl groups and the total protein solution volume.
3) vc-MMAE (dissolved in DMSO) was added to the reduced antibody at 1.1 times the moles of free sulfhydryl groups, mixed well, and then allowed to react with intermittent stirring at room temperature for 2 hours. To the reaction system, N-acetylcysteine was added to the reaction solution at 20 times the number of moles of vc-MMAE, mixed well and allowed to stand for 5 minutes.
4) The solution obtained in step 3 was replaced with the conjugate stock solution (10 mM histidine, 6% sucrose, 0.035% PS80, pH 5.8) using a 15 mL 30 KD ultrafiltration device for a total of 3 cycles. Substitution was performed to obtain antibody-drug conjugate ADC-1 stored at 4° C., the concentration of active ingredient antibody-drug conjugate was 5 mg/mL.

Example 4 Determination of Drug/Antibody Ratio in ADC-1 Prepared antibody-drug conjugate ADC-1 was analyzed by HIC-HPLC (Ouyang-2013) to determine the drug/antibody ratio (DAR) and The results are shown in Figure 1, in which the average drug loading DAR was 3.8 by calculation of the spectral peak areas.

Example 5 In Vitro Killing Activity of ADC-1 on CD20-Expressing NHL Cells To examine the killing effect of ADC-1 on CD20-expressing NHL cells, four CD20-expressing NHL cell lines Daudi, Jeko-1, Raji, and Ramos was selected and PrestoBlue™ detection reagent was employed to measure the killing effect of ADC-1 and the commercial reference drug Rituxan® on the above four cell lines and the results are shown in Table 2 and Figure 2, FIGS. 3, 4 and 5. FIG.

Note: References for CD20 expression levels were Barth-2015, Law-2004.
IC50 values are the mean of IC50 calculated for two 96-well plates, ND indicates the highest percentage of growth inhibition (% inhibition) was less than 50%, IC50 values could not be calculated.

Experimental results showed that ADC-1 had a significant cell-killing effect on four CD20-expressing NHL cell lines, and the effect was significantly better than the commercial reference drug Rituxan®. .

Example 6: Anti-tumor effect of ADC-1 in the NHL PDX model The human-derived tumor tissue xenograft model (PDX model) is a tumor model established in immunocompromised mice using human primary tumor tissue, and the primary tumor maximally preserve the heterogeneity, molecular diversity and histological features of It has high predictive value for the clinical therapeutic efficacy of drugs and has been used increasingly in cancer research in recent years (Hidalgo, 2014). To effectively evaluate the efficacy of ADC-1 in future clinical indications, experiments were performed in three CD20-expressing human lymphoma cell carcinoma (NHL) PDX models. In all experiments, the commercially available CD20-targeted monoclonal antibody Rituxan® was used as a reference drug to compare tumor suppressor activity. Of these three NHL PDX models, two were resistant to Rituxan®.

Information for the three PDX models used in this study is shown in Table 3.

1. Study of ADC-1 Efficacy in CD20-Positive NHL PDX Model LYM#004 LYM#004 is a Rituxan®-resistant DLBCL PDX model and the experimental results are shown in FIG. After administration of ADC-1 at doses of 1, 3 and 10 mg/kg, the T/C (%) on day 18 was 45.90% (P>0.05) and 4.46% (P<0.05), respectively. .001) and 2.05% (P<0.001), with TGI% of 54.10%, 95.54% and 97.95%, respectively. All tumors partially regressed on day 18 after administration of ADC-1 at a dose of 3 mg/kg, and 5/8 tumors partially regressed after administration of ADC-1 at a dose of 10 mg/kg. , and 3/8 tumors completely regressed on day 18, respectively. After 3 and 10 mg/kg Rituxan® administration, the T/C (%) on day 18 were 50.76% (P>0.05) and 51.50% (P>0.05), respectively. and the TGI% were 49.24% and 48.50%, respectively.

The experimental results showed that ADC-1 (3 and 10 mg/kg) had significant inhibitory activity against tumor growth, and ADC-1 (1 mg/kg) and the reference drug Rituxan® (3 and 10 mg/kg) ) did not show significant anti-tumor activity. Both ADC-1 and Rituxan® were well tolerated in tumor-bearing mice.

2. Examination of the efficacy of ADC-1 in the CD20-positive NHL PDX model LYM#013 LYM#013 is a Rituxan®-resistant DLBCL PDX model, and the experimental results are shown in FIG. After administration of ADC-1 at doses of 0.3, 1 and 3 mg/kg, the T/C (%) on day 28 were 31.90% (P<0.05) and 0.00% (P<0.05), respectively. <0.001), 0.00% (P<0.001); TGI% were 68.10%, 100.00%, 100.00%, respectively. 2/8, 0/8, and 0/8 tumors partially regressed, and 0/8, 8/8, and 8/8 tumors completely regressed, respectively. When Rituxan® was administered at 3 mg/kg, the T/C(%) on day 28 was 79.86% (P>0.05) and the TGI% was 20.14%.

Experimental results showed that ADC-1 (0.3, 1 and 3 mg/kg) could significantly inhibit tumor growth in a dose-dependent manner, whereas the control drug Rituxan® (3 mg/kg) significantly inhibited tumor growth. It did not show anti-tumor activity. Both ADC-1 and Rituxan® were well tolerated in tumor-bearing mice.

3. Study of ADC-1 Efficacy in CD20-Positive NHL PDX Model LYM#016 LYM#016 is a DLBCL PDX model and experimental results are shown in FIG. After administration of ADC-1 at doses of 0.3, 1 and 3 mg/kg, the T/C (%) on day 14 were 37.76% (P>0.05) and 4.27% (P>0.05), respectively. <0.01) and 1.91% (P<0.01), and the TGI% were 62.24%, 95.73% and 98.09%, respectively. All tumors partially regressed on day 14 after administration of ADC-1 at a dose of 1 mg/kg, and 6/8 tumors partially regressed after administration of ADC-1 at a dose of 3 mg/kg, respectively. 2/8 tumors regressed completely on the 14th day. When Rituxan® was administered at 3 mg/kg, T/C(%) was 30.98% (P>0.05) and TGI% was 69.02% on day 14.

Experimental results showed that ADC-1 (1 and 3 mg/kg) had a significant inhibitory effect on tumor growth, while ADC-1 (0.3 mg/kg) and the reference drug Rituxan (3 mg/kg) showed no significant anti-tumor activity. Both ADC-1 and Rituxan® were well tolerated in tumor-bearing mice.

Results of in vivo efficacy experiments of ADC-1 in three CD20-positive human lymphoma (NHL) PDX models are shown in Table 4.

Note: "/" stands for "not available". The formula for calculating tumor volume (TV) is TV=l×w2/2, where l and w represent the measured length and width of the tumor, respectively. Relative tumor volume (RTV) was calculated according to the measurement result RTV=Vf/V0. where V0 represents tumor volume measured at the time of grouping and dosing (ie, day 0) and Vf represents tumor volume measured on the last day. Relative tumor growth rate T/C (%) = (RTV in administration group/RTV in vehicle group) x 100%. Tumor growth inhibition rate TGI%=(average tumor volume of vehicle group−average tumor volume of administration group)/average tumor volume of vehicle group×100%. ++++ represents T/C (% 0 to 10%), "++++" represents T/C (% 10% to 20%), "++" represents T/C (% 20% to 40% % or less), "+" represents T/C (% or more than 40%). Partitioning of the anti-tumor activity of ADC-1 and Rituxan® was based on T/C (%) values at the highest dose in the model.

In conclusion, the results of in vivo efficacy experiments show that ADC-1 exhibits significant tumor growth inhibitory effects in three human lymphoma (NHL) PDX models (all models are DLBCL) and is very well tolerated in tumor-bearing mice. was shown to indicate It is worth pointing out that two of these three DLBCL PDX models were resistant to Rituxan®.

Example 7: Effect of pH on stability of ADC-1 formulations Gate (naked antibody-drug conjugate without buffers and other components), 10 mM histidine buffer (pH 5.8), 6% sucrose, and 0.035% Tween-80 (PS80). decisively decided.

To investigate the effect of different pH in 10 mM histidine buffer on the quality of ADC-1, the solutions of ADC-1 were changed from formulation F1 to F4 (pH: 5.8-7.0) for 6 weeks each. and 4 weeks at 25±2° C./60%±5% RH and 40±2° C./75%±5% RH for investigation and samples were taken for analysis at set time points. The detection items were appearance, pH, protein concentration, SEC, iCIEF and HIC. Subsequent association analysis is not performed if the sample is clearly emulsified. A specific experimental design is shown in Table 5.

Notes: W represents weeks, 2W represents 2 weeks, Check represents detected.

Experimental results showed that when ADC-1 is in 10 mM histidine buffer, its stability decreases with increasing pH.

After standing at 25°C for 6 weeks, all samples were colorless and clear solutions with no change in pH and concentration; the purity of SEC showed a decreasing trend, the polymer (HMW) content increased, and the rate of change increased with increasing pH; the DAR value did not change significantly.

After standing at 40°C for 4 weeks, the stability of the samples showed the same trend, but the degree of change was worse. At pH 6.7 and 7.0, the appearance of the sample changed within 1 week at 40°C and opalescence appeared, the degree of opalescence increased with increasing pH, and the presence of particulate matter caused UV The detection result was distorted (protein light). The calculated protein content deviated from the actual value. The purity of SEC decreased, the content of polymer and low molecular weight fragments (LMW) increased, and the rate of change increased with increasing pH. The purity of iCIEF decreased, the content of acidic peaks increased, the content of basic peaks decreased, and the rate of change increased with increasing pH.

A trend plot of the SEC purity of the samples as a function of pH is shown in FIG.

Although specific embodiments of the invention have been described in detail, various modifications and substitutions of these details can be made by those skilled in the art in light of the full teachings disclosed, and these changes will make the present invention a reality. will be understood to be within the range of The full scope of the invention is given by the appended claims and their equivalents.

Claims (13)

Formula I:
Ab-(LD) _p Formula I
An antibody-drug conjugate having the structure shown in
Ab stands for anti-CD20 monoclonal antibody, anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or its biosimilars;
D is a cytotoxic agent, the cytotoxic agent is monomethylauristatin E (MMAE);
L represents a linker for linking the anti-CD20 monoclonal antibody with the cytotoxic agent, the linker being 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB);
The above antibody-drug conjugate, wherein p is 3.6-4.0, preferably 3.7-3.9, more preferably 3.8. An antibody-drug conjugate preparation comprising:
an antibody-drug conjugate having a concentration of 1-60 mg/mL (e.g., a concentration of 1-20 mg/mL, or 1-10 mg/mL, or 3-7 mg/mL, or 5 mg/mL);
a histidine buffer with a concentration of 5-35 mM (eg, a histidine buffer with a concentration of 5-15 mM, or 8-12 mM, or 10 mM) and a pH of 5.0-6.2 (eg, 5.8-10 mM); pH of 6.2, or pH of 5.4-6.0, or pH of 5.4-6.2, or pH of 5.6-6.0);
sucrose at a concentration of 2% to 10% (e.g. sucrose at a concentration of 3% to 9%, or 4% to 8%, or 5% to 7%, or 6%);
Tween-80 at a concentration of 0.01% to 0.1% (e.g., at a concentration of 0.02% to 0.06%, 0.02% to 0.05%, 0.03% to 0.04% or Tween-80 which is 0.035%);
The antibody-drug conjugate has Formula II:
Ab-(LD)q Formula II:
having the structure shown in
Ab stands for anti-CD20 monoclonal antibody, anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or its biosimilars;
D is a cytotoxic agent, the cytotoxic agent is monomethylauristatin E (MMAE);
L represents a linker for linking the anti-CD20 monoclonal antibody with the cytotoxic agent, the linker being 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB);
q is 3.3-4.3 (e.g., 3.6-4.0, 3.7-3.9, 3.8);
Preferably, the preparation is
an antibody-drug conjugate having a concentration of 5 mg/mL;
10 mM histidine buffer at pH 5.8;
6% sucrose; and 0.035% Tween-80
The above antibody-drug conjugate preparation comprising: A method for preparing an antibody-drug conjugate preparation as defined in claim 2, comprising:
Step 1: A step of reducing anti-CD20 monoclonal antibody and a reducing agent to obtain a reduced anti-CD20 monoclonal antibody, wherein the anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or its biosimilars a process that is
Step 2: performing a coupling reaction between the reduced anti-CD20 monoclonal antibody and vc-MMAE;
Step 3: Stopping the coupling reaction;
Step 4: Buffer replacement of the quenched coupling reaction product to obtain an antibody-drug conjugate preparation, the antibody-drug conjugate preparation comprising:
1-60 mg/mL antibody-drug conjugate (eg, 1-20 mg/mL, or 1-10 mg/mL, or 3-7 mg/mL, or 5 mg/mL antibody-drug conjugate);
5-35 mM histidine buffer (eg, 5-15 mM, or 8-12 mM, or 10 mM histidine buffer), pH 5.0-6.2 (eg, pH 5.8-6.2, or pH 5.4- 6.0, or pH 5.4-6.2, or pH 5.6-6.0);
2-10% sucrose (eg 3-9%, 4-8%, 5-7%, 6% sucrose); and 0.01%-0.1% Tween-80 (eg 0.02% ~0.06%, 0.02%-0.05%, 0.03%-0.04%, or 0.035% Tween-80).
The above preparation method, comprising a step comprising said method comprising:
1) Collect 10 mg of anti-CD20 monoclonal antibody and replace with reducing buffer (25 mM sodium borate, pH 8.0, 25 mM NaCl, 5 mM EDTA) using a 15 mL 30 KD ultrafiltration device for a total of 3 replacements. to obtain a final volume of approximately 1 mL, transfer the resulting solution to a new Eppendorf centrifuge tube (weigh), detect protein concentration and calculate total protein;
2) DTT was added to the antibody at 2.0-3.0 fold molar, incubated for 2 hours at room temperature, mixed continuously, and then filtered using 15 mL of 30 KD ultrafiltration device with coupling buffer (50 mM Tris , pH 7.2, 150 mM NaCl, 5 mM EDTA), performing 3 substitutions, collecting the concentrate, detecting protein concentration at A280 (absorbance at wavelength 280 nm), weighing, weighing total protein content. , collect 10 μL of the sample, and determine the number of free sulfhydryl groups by Ellman's method;
The molar concentration of free sulfhydryl groups is given by the formula:

is calculated by
b: optical path length of the cuvette (usually 1 cm)
The number of moles of free sulfhydryl groups is calculated according to the molar concentration of free sulfhydryl groups and the volume of the total protein solution;
3) vc-MMAE (dissolved in DMSO) is added to the reduced antibody at 1.0-1.5 times the number of moles of free sulfhydryl groups, mixed well, then at room temperature. React for 2 hours with intermittent stirring; Add N-acetylcysteine to the reaction system at 20 times the number of moles of vc-MMAE, mix well, and stand for 5 minutes;
4) substituting the solution obtained in step 3 with the conjugate stock solution (10 mM histidine, 6% sucrose, 0.035% PS80, pH 5.8) using 15 mL of a 30 KD ultrafiltration device for a total of 3 substitutions; to obtain an antibody-drug conjugate preparation and store at 4°C;
Preferably, the preparation method comprises
1) Collect 10 mg of anti-CD20 monoclonal antibody and replace with reducing buffer (25 mM sodium borate, pH 8.0, 25 mM NaCl, 5 mM EDTA) using a 15 mL 30 KD ultrafiltration device for a total of 3 replacements. to obtain a final volume of approximately 1 mL, transfer the resulting solution to a new Eppendorf centrifuge tube (weigh), detect protein concentration and calculate total protein;
2) DTT was added to the antibody at 2.5-fold molar, incubated at room temperature for 2 hours, and continuously mixed, followed by coupling buffer (50 mM Tris, pH 7.2, 150 mM NaCl, 5 mM EDTA), substituted three times, collected the concentrate, detected the protein concentration by A280, determined the total protein amount, collected a 10 μL sample, and determined the number of free sulfhydryl groups by Ellman's method. seek;
The molar concentration of free sulfhydryl groups is given by the formula:

is calculated by
b: optical path length of the cuvette (usually 1 cm)
The number of moles of free sulfhydryl groups is calculated according to the molar concentration of free sulfhydryl groups and the volume of the total protein solution;
3) vc-MMAE (dissolved in DMSO) is added to the reduced antibody at 1.1 times the number of moles of free sulfhydryl groups and mixed well, then with intermittent stirring at room temperature for 2 hours. react; add N-acetylcysteine to the reaction system at 20 times the number of moles of vc-MMAE, mix well, and leave for 5 minutes;
4) Using a 15 mL 30 KD ultrafiltration device, the solution obtained in step 3 was replaced with the conjugate stock solution (10 mM histidine, 6% sucrose, 0.035% PS80, pH 5.8), and filtered three times in total. 4. The preparation method according to claim 3, comprising performing the replacement to obtain the antibody-drug conjugate preparation and storing at 4° C., the concentration of the active ingredient antibody-drug conjugate being 5 mg/mL. An antibody-drug conjugate as defined in claim 1, or an antibody-drug conjugate preparation as defined in claim 2, or an antibody prepared by the method as defined in any one of claims 3-4. - A composition comprising a drug conjugate preparation, optionally further comprising at least one pharmaceutically acceptable carrier, diluent or excipient. The antibody-drug conjugate of claim 1, or the antibody-drug conjugate preparation of claim 2, or the antibody-drug conjugate preparation of claim 3, in the manufacture of a medicament for the prevention and/or treatment of a CD20-expressing cancer or immune disease. Use of the antibody-drug conjugate preparation prepared by the method of any one of claims 1-4 or the composition of claim 5. CD20-expressing cancer or immune disease is lymphoma (e.g., non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, small lymphocytic lymphoma, diffuse large B-cell lymphoma), leukemia (e.g., chronic lymphocytic leukemia, hairy cell leukemia, B-cell prolymphocytic leukemia, acute lymphocytic leukemia), rheumatoid arthritis (e.g., severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist), polyangiitis associated granulomatosis, Wegener's granulomatosis, microscopic polyangiitis or multiple sclerosis;
Preferably, the CD20-expressing cancer is a CD20-positive B-cell lymphoma, preferably an anti-CD20 monoclonal antibody (e.g., rituximab) resistant CD20-positive B-cell lymphoma;
More preferably, the lymphoma is non-Hodgkin's lymphoma, preferably anti-CD20 monoclonal antibody (e.g., rituximab) resistant non-Hodgkin's lymphoma,
More preferably, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma, preferably anti-CD20 monoclonal antibody (eg rituximab) resistant diffuse large B-cell lymphoma. A method for the prevention and/or treatment of a CD20-expressing cancer or immune disease, comprising a prophylactically and/or therapeutically effective amount of an antibody-drug conjugate as defined in claim 1 or as defined in claim 2. or an antibody-drug conjugate preparation prepared by the method defined in any one of claims 3-4, or a composition as defined in claim 5. A method comprising administering to a subject. CD20-expressing cancer or immune disease is lymphoma (e.g., non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, small lymphocytic lymphoma, diffuse large B-cell lymphoma), leukemia (e.g., chronic lymphocytic leukemia, hairy cell leukemia, B-cell prolymphocytic leukemia, acute lymphocytic leukemia), rheumatoid arthritis (e.g., severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist), polyangiitis associated granulomatosis, Wegener's granulomatosis, microscopic polyangiitis or multiple sclerosis;
Preferably, the CD20-expressing cancer is a CD20-positive B-cell lymphoma, preferably an anti-CD20 monoclonal antibody (e.g., rituximab) resistant CD20-positive B-cell lymphoma More preferably, the lymphoma is a non-Hodgkin's lymphoma, preferably an anti-CD20 monoclonal Antibody (e.g., rituximab)-resistant non-Hodgkin lymphoma,
More preferably, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma, preferably anti-CD20 monoclonal antibody (eg, rituximab) resistant diffuse large B-cell lymphoma. When the subject is human, the dose administered to a subject in need thereof is 0.1-5 mg/kg (e.g., 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg);
Alternatively, if the subject is an NHL PDX model, the dose administered to a subject in need thereof is 0.3-10 mg/kg (eg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg). kg), preferably 0.3-3 mg/kg, 1-3 mg/kg, 1-10 mg/kg, or 3-10 mg/kg. for use in the prevention and/or treatment of CD20-expressing cancers or immune diseases, an antibody-drug conjugate as defined in claim 1 or an antibody-drug conjugate preparation as defined in claim 2, or An antibody-drug conjugate preparation prepared by a method as defined in any one of claims 3-4, or a composition as defined in claim 5. CD20-expressing cancer or immune disease is lymphoma (e.g., non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, small lymphocytic lymphoma, diffuse large B-cell lymphoma), leukemia (e.g., chronic lymphocytic leukemia, hairy cell leukemia, B-cell prolymphocytic leukemia, acute lymphocytic leukemia), rheumatoid arthritis (e.g., severe active rheumatoid arthritis that has failed treatment with at least one TNF antagonist), polyangiitis associated granulomatosis, Wegener's granulomatosis, microscopic polyangiitis or multiple sclerosis;
Preferably, the CD20-expressing cancer is a CD20-positive B-cell lymphoma, preferably an anti-CD20 monoclonal antibody (e.g., rituximab) resistant CD20-positive B-cell lymphoma;
More preferably, the lymphoma is non-Hodgkin's lymphoma, preferably anti-CD20 monoclonal antibody (e.g. rituximab) resistant non-Hodgkin's lymphoma;
More preferably, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma, preferably anti-CD20 monoclonal antibody (e.g. rituximab) resistant diffuse large B-cell lymphoma. antibody-drug conjugate or antibody-drug conjugate preparation or composition for When the subject is human, the dose administered is 0.1-5 mg/kg (e.g., 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg);
Alternatively, if the subject is an NHL PDX model, the dose administered is 0.3-10 mg/kg (eg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg), preferably 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg. The antibody-drug conjugate or antibody-drug conjugate for use as defined in claim 11, which is 3-3 mg/kg, 1-3 mg/kg, 1-10 mg/kg, or 3-10 mg/kg. preparation or composition.

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